1. Area of the Art
The invention relates generally to activated dyes and specifically to activated cyclic-bridged cyanine dyes, their synthesis and methods of use in labeling of biopolymers.
2. Description of the Prior Art
Many procedures employed in biomedical research and recombinant DNA technology rely heavily on the use of nucleotide or polynucleotide derivatives radioactively labeled with isotopes. However, the rapidly increasing costs of radioactive waste disposal, together with an increased awareness of the potentially harmful effects of exposure to radiation, have contributed to a shift of emphasis toward other ways of labeling synthetic oligonucleotides. Although many different types of non-radioactive labels have found their use in biological detection assays, use of fluorescent labels has expanded rapidly in recent years due to both improvements in detection instrumentation and the increased number of novel fluorescent labeling reagents.
The sensitivity and accuracy of fluorescence detection techniques depend on the physical and chemical characteristics of the dyes they employ. A common problem with many commercially available fluorescent labeling reagents is that they are not water-soluble and must be dissolved in organic solvents prior to labeling substrate in aqueous media. Such organic solvents can have a deleterious effect upon sensitive substrates. Another problem related to the dye""s chemical structure is non-specific staining of cellular matter by the dye, which reduces signal-to-noise ratio during observation.
Cyanine and related dyes offer many advantages over existing fluorescent labeling reagents, including a high extinction coefficient, relatively high quantum efficiency, ease of chemical manipulation, and reasonable stability to reagents, pH and temperature. Because of a low fluorescence background of biological materials and a high absorbency of cyanine dyes in the longer wavelength portion of the spectrum, cyanine dyes provide excellent signal-to-noise ratios. Certain cyanine and related dyes are relatively photo-stable and do not rapidly bleach under the fluorescence microscope. They can be covalently attached to biological and non-biological markers to make these materials fluorescent. Additionally, due to their relatively small size, cyanine dyes minimally perturb the function of the labeled product. Finally, the versatility of functional groups that can be incorporated into cyanine dyes permits control over the solubility of the dye and labeled product and helps reduce non-specific binding of the labeled materials to irrelevant components in an assay mixture (Waggoner, U.S. Pat. No. 5,569,587 and U.S. Pat. No. 5,627,027).
In order to improve covalent attachment of cyanine dyes to target molecules, techniques for activating cyanine dyes by the incorporation of a reactive functional group (or activating group) have been developed. Waggoner (U.S. Pat. No. 5,569,587 and U.S. Pat. No. 5,627,027) has presented numerous cyanine dye derivatives that can be used as covalently reacting molecules. The reactive groups used in these dyes are isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono-or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, aziridine, sulfonyl halide, acid halide (except for fluorides), hydroxysuccinimide ester, hydroxy sulfosuccinimide ester, imido ester, glyoxal and aldehyde. Waggoner has suggested incorporation of carboxylic groups into the basic cyanine structure to increase solubility of the dye in water and to permit fluorescent labeling through the use of derived active esters (U.S. Pat. No. 4,981,977 and U.S. Pat. No. 5,627,027).
Cyanine dyes have a general structure where the chromophore of the cyanine dyes is composed of a series of conjugated double bonds having two quaternary nitrogen atoms at the terminal ends which share one positive charge. According to the number of central double bonds, the cyanine dyes can be classified as monocarbocyanine (also known as trimethinecarbocyanine or Cy3), dicarbocyanine (also known as pentamethinecarbocyanine or Cy5), and tricarbocyanine (also known as heptamethinecarbocyanine or Cy7). The number of central double bonds (referred to hereinafter as k) determines in part the excitation color. Often, higher values of k contribute to increased fluorescence and absorbance. At values of k above 4, however, the compound becomes unstable. Thereupon, further fluorescence can be imparted by modifications at the ring structures. When k=2, the excitation wavelength is about 650 nm and the compound is very fluorescent.
By synthesizing structural modifications of the chromophore portion of cyanine dyes, different fluorescent labeling reagents absorbing and emitting in a broad spectrum range from 400 to nearly 1100 nm can be obtained. For example, U.S. Pat. No. 5,571,388 describes pentamethine and heptamethine cyanine dyes incorporating various cyclic structures within a chain of conjugated double bonds. These dyes absorb light having wavelengths from 630 nm to 900 nm.
Although above-described efforts have increased the number of cyanine dyes suitable for labeling biomolecules, many of these dyes are not especially photostable, and their solubility properties are not optimal for many uses that would involve fluorescence detection of labeled materials. It would be highly desirable to provide stable cyanine dyes having suitable absorption and fluorescence properties and useful linking groups for attachment to biomolecules. It is also desirable to have dyes that can be used with both organic and aqueous solvents.
We have discovered that dyes having more than two central double bonds (for example, Cy7 dyes) are particularly unstable during storage and manipulation. Accordingly, it is an object of the present invention to provide stable fluorescent dyes with more than two central double bonds and stable Cy7 dyes, in particular. It is also an object of the present invention to provide convenient methods for their synthesis and use in labeling biological and non-biological materials.
These and other objects are achieved in dyes of the present invention having a following general formula: 
In this formula, each dotted line represents carbon atoms necessary to form a fused substituted or unsubstituted aromatic ring; n=1-18; and m=1-18, selected independently from n. X and Y are independently selected from the group consisting of S, O, N, CH2 and C(CH3)2, and at least one of R1 and R2 comprises a sulfonic acid or sulfonate group attached to the aromatic ring. R3 and R4 are independently selected from the group consisting of carboxyl, activated carboxyl and methyl. According to the present invention, it is required that at least one of said R3 and R4 groups is carboxylate or activated carboxylate. In one embodiment, both R3 and R4 are a carboxylate or an activated carboxylate. In another embodiment, either R3 or R4 is carboxylate or activated carboxylate. The activated carboxylate may be an ester, for example an ester of N-hydroxynapthalimide. According to embodiments of the present invention, the dyes may be cyclic-bridged cyanine dyes and, more particularly, cyclic-bridged Cy7-type cyanine dyes.
Another aspect of the present invention provides a method of synthesizing a cyclic-bridged dye. The method includes the steps of:
(a) forming a cyclic-bridged derivative of the dye having a formula: 
xe2x80x83(b) replacing the halogen with a hydrogen.
In the formula (II), each dotted line represents carbon atoms necessary to form a fused substituted or unsubstituted aromatic ring; n=1-18; m=1-18, selected independently from n; and Z is a halogen. X and Y are selected independently from the group consisting of S, O, N, CH2 and C(CH3)2 and at least one of the R1 and R2 comprises a sulfonic acid or sulfonate group attached to the aromatic ring. R5 and R6 are independently selected from a carboxyl or a methyl, wherein at least one of said R5 and R6 is a carboxyl.
According to embodiments of the present invention, the dye can be a cyanine dye, and particularly Cy7, BCy7, and DBCy7. In one embodiment, both R5 and R6 are carboxyls. In another embodiment, either R5 or R6 is carboxyl. The halogen may be a chlorine.
In one embodiment of the present invention, the step of forming a cyclic-bridged derivative of the dye comprises the steps of:
(a1) mixing Compounds (XI) and (XII) with 2-chloro-1-formyl-3-hydroxymethylene-cyclohexene to form a reaction mixture; and
(a2) maintaining the reaction mixture under conditions that allow the formation of the cyclic-bridged derivative of the dye.
Compound (XI) may be any compound having a general formula: 
Compound (XII) may be any compound having a general formula: 
In the formulas of Compounds (XI) and (XII), each dotted line represents carbon atoms necessary to form a fused substituted or unsubstituted aromatic ring; n=1-18; m=1-18, selected independently from n; and X and Y are selected from the group consisting of S, O, N, CH2 and C(CH3)2. At least one of the groups R1 and R2 comprises a sulfonic acid or sulfonate group attached to the aromatic ring. Groups R5 and R6 are independently selected from a carboxyl or a methyl, wherein at least one of said R5 and R6 is carboxyl.
Another aspect of the present invention provides a method wherein the activated cyclic-bridged dye of this invention is used to label a biological or a non-biological material.
The present invention has been found to provide a number of advantages. By incorporating a cyclic moiety into the chain of conjugated double bonds of Cy7-type dyes, their partial conversion into Cy5-like species is effectively prevented and stable dyes are formed. As a result, a higher overall yield of labeled product is achieved as compared to labeling with unmodified Cy7-type dyes. As explained in greater detail below, N-hydroxynaphthalimide ester of Cyclic DBCy7 was successfully conjugated to amino oligonucleotides and dideoxynucleotides. While cyclic-bridged DBCy7-labeled terminators performed well in sequencing, cyclic-bridged DBCy7-labeled primers produced good results in DNA Fragment Analysis. Many other procedures employed in biomedical research and using fluorescent dyes may also benefit from the dyes of the present invention.
The invention is defined in its fullest scope in the appended claims and is described below in its preferred embodiments.